This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. Alpha-Dystroglycan ([unreadable]DG) possesses a mucin-like domain with multiple serine (S) or threonine (T) residues with O-linked mannosylated (O-Man) oligosaccharides. These O-Man moieties can then be elongated by the O-mannosyl-[unreadable]1,2-N- acetylglucosaminyltransferase (POMGnT1) and by a series of glycosyltransferases. Recent reports have shown that mutations in POMGnT1 are a major cause of a form of muscular dystrophy known as muscle-eye-brain (MEB) disease. In order to gain a better understanding of the possible role of POMGnT1 in the modifications of [unreadable]DG that lead to MEB disease, we have synthesized eight peptide sequences derived from the mucin-like domain of [unreadable]DG, each with one or multiple O-Man sites. These peptides were designated as M1 (residues 416-420 with one O-Man at T418), M2 (residues 429-433 with two O-Man sites at S430 and T431), M3 (residues 326-331 with two O-man sites at T328 and T329), M4 (residues 411-416 with an O-Man at T414), M5 (residues 461-466 with two O-Man sites at T463 and T464), M6 (residues 480-487 with four O-Man sites at T482, T483, T484, and S485), M7 (residues 419-427 with two O-Man sites at T421 and T424), and M8 (residues 419-427 with four O-Man sites at T421, T422, T423, and T424). These peptides are being used as in vitro acceptors for recombinant POMGnT1 expressed in Human embryonic kidney (HEK-293) cells. In addition to the kinetic parameters (Km, Kcat, and Km/Kcat) of this enzyme for each substrate, MSn fragmentation experiments are being performed in the reaction products to determine whether POMGnT1 has a preference for the addition of GlcNAC to specific O-mannosylated sites, or both sites are affected by the glucosaminyltransferase. In cases where both sites are affected by POMGnT1, in order to determine whether the action of POMGnT1 is sequential, or the addition of GlcNAc to the O- mannosylated residues occurs randomly, time course enzymatic reaction experiments are conducted in which the products are analyzed by MS to determine to which mannosylated sites the GlcNac residues are being added.